Porcine Thyroid Epithelial Cells Research Articles

SUN-LB129 Thyroglobulin Stimulates the Secretion of Thyroxine From Thyroid Epithelial Cells in Suspension Culture via Nuclear Factor-κB Signaling Pathway

The nuclear factor (NF)-κB signaling pathway controls a variety of biological functions such as cell growth and differentiation as well as immune and inflammatory responses. Two distinct pathways of NF-κB activation are known. NF-κB signaling via non-canonical pathway plays an important role in maintaining normal thyroid function, namely, thyroid cells survival and expression of thyroid-specific proteins, such as NIS, TPO and thyroglobulin (Tg). The primary function of thyroid cells is the production of thyroid hormones, which are cleaved out by various processing enzymes from the modified tyrosine residues of the Tg molecule. Although Tg is currently recognized as no more than a scaffold protein for thyroid hormone synthesis, our group has shown that Tg stimulates the proliferation of rat thyroid follicular FRTL-5 cells via PI3K pathway. The signaling pathways underlying various functions of thyroid cells have been extensively investigated but some discrepancies still exist, depending on the experimental model systems and the types of cells used. Recently, our group discovered that Tg may directly regulate the synthesis of thyroid hormones in thyroid cells. In this study, we performed suspension culture of porcine thyroid cells and aimed to elucidate the mechanism of the effect of Tg on thyroid cells. Porcine thyroid epithelial cells were prepared from thyroid gland by enzymatic digestion and cultured in monolayer or in suspension culture. Various concentrations of Tg were added to the medium and cultured for several days. Then, the conditioned medium was collected and the amount of thyroid hormone was measured by reversed phase chromatography; the cells were harvested and mRNA levels of hormone-processing enzymes, cathepsin B, K, L and dipeptidyl peptidase I, II (DPP I, II), were measured by quantitative PCR. The amount and activation of cell signaling molecules were also evaluated by western blotting. Tg stimulated the secretion of thyroid hormone, thyroxine, from porcine thyroid cells in suspension culture, however, no stimulation by Tg was observed in monolayer culture. The expression of mRNAs of cathepsin B, K and L were reduced and that of DPP II was increased by the treatment of Tg in suspension culture. Tg increased the amount of NF-κB p52 and Rel B dose dependently. On the other hand, TSH had little effects on the amount of these proteins. Tg and TSH both activated PDK1 and MAPK p44/42 but not Akt. These results suggest that Tg regulates thyroid hormone synthesis in totally different way from TSH by altering the expression levels of hormone-processing enzymes in thyroid cells. This stimulatory effect might be mediated by NF-κB signaling pathway, whereas the proliferative effect of Tg under these conditions might be exerted via MAPK signaling pathway. The detailed mechanisms of these effects of Tg on thyroid cells are under investigation.

Proliferative Effects of Bovine and Porcine Thyroglobulins on Thyroid Epithelial Cells

Thyroglobulin is the precursor of the thyroid hormones, triiodothyronine and thyroxine. Because the molecular size of thyroglobulin is relatively large (660 kDa), it could have other additional functions besides serving as the precursor of the thyroid hormones. In this report, we examined the proliferative effects of thyroglobulins purified from bovine and porcine thyroid tissues on the growth of a rat thyroid follicular cell line, FRTL-5, as well as the primary culture of porcine thyroid epithelial cells. Bovine and porcine thyroglobulins stimulated the proliferation of not only FRTL-5 cells but also porcine thyroid epithelial cells in a dose-dependent manner. The proliferative effect of thyroglobulin was neutralized by an anti-thyroglobulin monoclonal antibody but not by two different anti-fibroblast growth factor antibodies. The stimulatory signal of thyroglobulin was transmitted via the phosphatidylinositol 3-kinase pathway. Also, removal of the N-linked oligosaccharides on thyroglobulin reduced the proliferative activity of porcine thyroglobulin, suggesting that the proliferative effect of thyroglobulin is in part exerted by its carbohydrate moiety. Taken together, we have demonstrated for the first time that thyroglobulin possesses proliferative effect on thyroid epithelial cells in addition to being the precursor of the thyroid hormones.

Similarities and differences between free 211At and 125I− transport in porcine thyroid epithelial cells cultured in bicameral chambers

The transport and accumulation of free 211At and 125I− were investigated in thyrocytes cultured as monolayers in bicameral chambers under the influence of thyroid-stimulating hormone, stable iodide, ouabain and perchlorate. The results indicate that there are similarities and differences in the transport mechanisms of free 211At and 125I−. These results will be valuable in the development of radiation protection when handling and using 211At-labeled radiopharmaceuticals, and for the potential use of free 211At in radiation therapy of poorly differentiated thyroid cancer.

UTP-preferring P2 receptor mediates inhibition of sodium transport in porcine thyroid epithelial cells.

1. The effects of adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP) and analogues on forskolin-stimulated absorption of Na+ by porcine thyroid epithelial cells were analysed in cultures grown as confluent monolayers on permeable supports in Transwell Ussing chambers. 2. 85% of the forskolin (10 microM)-stimulated short-circuit current was inhibited by phenamil (1 microM), which is a selective antagonist for epithelial type Na+ channels. 3. Phenamil-sensitive current was inhibited in a dose dependent manner by nucleotides added to the apical compartment of Ussing chambers. In contrast, the phenamil-resistant current, previously shown to represent anion secretion, was unaffected by nucleotides. 4. The order of potency (with EC50 values given in microM) was UTP (0.08)>>ATP (6.3)=uridine 5'-diphosphate (UDP) (6. 6)>2methyl-thio-adenosine-5'-triphosphate (2MeSATP) (84.5)>adenosine 5'-diphosphate (ADP) (147.8)>alpha,beta-methylene ATP (>150)>>adenosine (>1000). 5. P2 receptors mediating inhibition of sodium absorption were present on the apical membrane of the cells since addition of UTP (1-1000 microM) to the basal compartment of the Ussing chambers had little effect while subsequent addition to the apical compartment produced a normal response. 6. Cibachron blue (Reactive blue 2) (1-100 microM), an antagonist at some P2 receptor subtypes, inhibited phenamil sensitive current in a dose dependent manner with half maximal inhibition occurring at 14.25 microM. 7. Suramin (100 microM), pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS) (100 microM) and pyridoxal 5'-phosphate (P5P) (100 microM) showed only slight competitive antagonism against the response to UTP. 8 These results indicate that a UTP-preferring P2 receptor located on the apical membrane of thyroid epithelial cells mediates inhibition of Na+ absorption.

Influence of transforming growth factor beta1 (TGF-beta1) on the behaviour of porcine thyroid epithelial cells in primary culture through thrombospondin-1 synthesis.

Transforming growth factor beta1 (TGF-beta1) is a secreted polypeptide that is thought to play a major role in the regulation of folliculogenesis and differentiation of thyroid cells. On porcine thyroid follicular cells cultured on plastic substratum, TGF-beta1, in a concentration-dependent way, promoted the disruption of follicles, cell spreading, migration and confluency by a mechanism that did not involve cell proliferation. TGF-beta1 strongly activated the production of thrombospondin-1 and (alpha)vbeta3 integrin in a concentration-dependent manner whereas the expression of thyroglobulin was unaffected. Anisomycin, an inhibitor of protein synthesis, inhibited the effect of TGF-beta1 on cell organization. Thrombospondin-1 reproduced the effect of TGF-beta1. In the presence of thrombospondin-1 cells did not organize in follicle-like structures but, in contrast, spreaded and reached confluency independently of cell proliferation. This effect is suppressed by an RGD-containing peptide. The adhesive properties of thrombospondin-1 for thyroid cells were shown to be mediated by both the amino-terminal heparin-binding domain and the RGD domain of thrombospondin-1. Adhesion was shown to involve (alpha)vbeta3 integrin. The results show that TGF-beta1 exerted an influence upon function and behaviour of follicle cells partly mediated by the synthesis of thrombospondin-1 and of its receptor (alpha)vbeta3 integrin.

Polarized targeting of epithelial cell proteins in thyrocytes and MDCK cells.

Polarized trafficking signals may be interpreted differently in different cell types. In this study, we have compared the polarized trafficking of different proteins expressed endogenously in primary porcine thyroid epithelial cells to similar proteins expressed in MDCK cells. As in MDCK cells, NH4Cl treatment of filter-grown thyrocytes caused mis-sorted soluble proteins to exhibit enhanced secretion to the apical medium. In independent studies, thrombospondin 1 (a thyroid basolaterally secreted protein) was secreted basolaterally from MDCK cells. Likewise, the 5'-deiodinase (a thyroid basolateral membrane protein) encoded by the DIO1 gene was also distributed basolaterally in transfected MDCK cells. Consistent with previous reports, when the secretion of human growth hormone (an unglycosylated regulated secretory protein) was examined from transfected MDCK cells, the release was nonpolarized. However, transfected thyrocytes secreted growth hormone apically in a manner dependent upon zinc addition. Moreover, two additional regulated secretory proteins expressed in thyrocytes, thyroglobulin (the major endogenous glycoprotein) and parathyroid hormone (an unglycosylated protein expressed transiently), were secreted apically even in the absence of zinc. We hypothesize that while cellular mechanisms for interpreting polarity signals are generally similar between thyrocytes and MDCK cells, thyrocytes allow for specialized packaging of regulated secretory proteins for apical delivery, which does not require glycosylation but may involve availability of certain ions as well as appropriate intracellular compartmentation.

Sodium channel heterogeneity in the apical membrane of porcine thyroid epithelial cells

Porcine thyroid epithelial cells cultured as a monolayer with their apical membranes facing the medium are known to absorb Na+ and secrete Cl-. Two types of Na+ channels were found in cell-attached patches of apical membrane. A low conductance Na+ channel (conductance g = 4 picosiemens (pS)) remained open for seconds and showed a high selectivity for Na+ compared with K+. In contrast, a high conductance Na+ channel (g = 10 pS) flickered rapidly and had reduced selectivity. Both types of Na+ channel became more prevalent when the cells were exposed to Na(+)-free medium, though only the high conductance channel increased in prevalence on addition of prostaglandin E2, a stimulator of adenylate cyclase which increases Na+ absorption in this cultured epithelium. Two minority types of channel were also found: a non-selective small conductance cation channel which had been reported previously, and an intermediate conductance channel found only in Na(+)-free medium. It was concluded that passage of Na+ across the apical membrane of thyroid cells is mediated by typical epithelial Na+ channels, but that the two types of channel are differentially regulated.

Effects of Epidermal Growth Factor and Phorbol Ester on Thyroid Epithelial Integrity

The effects of epidermal growth factor (EGF) and phorbol ester (tetra- O-decanoylphorbol-16-acetate; TPA) on thyroid epithelial integrity were studied in filter-cultured monolayers of porcine thyrocytes, which before experiments were growth-arrested and had a high transepithelial resistance ( R TE > 6 · 10 3 Ω · cm 2) and polarized, thyroid-specific functions. Both EGF and TPA stimulated dose-dependently the cellular incorporation of [ 3H]thymidine, which maximally (at 10 ng/ml EGF for 48 h) corresponded to a 65% increase of the DNA content. The EGF-treated cells proliferated mainly within the original monolayer, which became folded due to the increased cell number; clusters of epithelial cells also assembled between the monolayer and the filter. Although the transepithelial potential difference was reduced, from 15-30 mV in controls to 2-10 mV, the epithelial barrier function was maintained ( R TE 1-3 · 10 3 Ω · cm 2; impermeability to [ 3H]inulin). EGF did not change the ultrastructural polarity of the plasma membrane or the distinct distribution of ZO-1 and cadherin immunoreactivities to junctions, but cytoplasmic cadherin present in controls disappeared after EGF. In cultures acutely depleted of extracellular Ca 2+ EGF pretreatment for 48 h antagonized the preventive effect of thyrotropin on paracellular leakage and loss of cell-cell adhesion. TPA (0.1 μ M) induced a temporary barrier dysfunction (maximal after 24 h) accompanied by pronounced alterations of cell shape and actin-based cytoskeleton, dissociation of junctional cadherin, and shedding of cells into the apical medium. In long-term (2-5 days) TPA-treated cultures the epithelial morphotype and barrier function recovered. The combined stimulation with EGF and TPA caused a persistent derangement of the cell layer including attenuation of ZO-1 at cell-cell contacts, paracellular leakage of [ 3H]inulin, and cell detachment. We conclude that EGF is able to release porcine thyroid epithelial cells from contact inhibition of growth along with intact cell polarity and tight junctions. Yet, when acting together with phorbol ester EGF provokes a lasting morphological transformation. Impaired positive control of Ca 2+-dependent cell-cell adhesion in EGF-treated cultures suggests a latent defect with possible transforming potential in the cadherin-based regulation of the junctional complex.

Effects of epidermal growth factor on basolateral iodide uptake and apical iodide permeability in filter-cultured thyroid epithelium.

The effect of epidermal growth factor (EGF) on vectorial iodide (I-) transport was studied in an in vitro model of the polarized porcine thyroid follicular epithelium in Transwell culture chambers. In this model, two mechanisms responsible for the unidirectional transport of I- from the basal to the apical chamber compartments, corresponding to the interstitium to lumen direction in vivo, are present in opposite plasma membrane domains of the tight cell monolayer: a basolateral I- pump and an apical I- efflux mechanism, both regulated by TSH. The cultures were treated with EGF (10 ng/ml) with or without TSH (0.1 mU/ml) for 48 h, and then analyzed for 1) cellular uptake of 125I-, 2) transepithelial 125I- flux (FTE 125I-; basoapical and apicobasal directions) during continuous exposure to 125I- in either the basal or apical medium, and 3) bidirectional (apical and basal) efflux in 125I(-)-loaded cells. Iodination was prevented by methimazole (0.5 mM). EGF increased cell number 25-50%, but did not disturb the tightness or structural polarity of the original cell monolayer. EGF reduced the basal rate and inhibited the TSH-induced long term up-regulation of FTE 125I- across the cell layer in the basoapical direction. However, the radioactivity content of EGF-treated cultures exposed to 125I- in the basal medium was 3-5 times higher than that in controls; apical uptake of 125I- was negligible. The radioiodide accumulated in EGF-treated cells was predominantly released (approximately 80%) in the basal direction, whereas in controls, the ratio of apical to basal 125I- efflux was 3:2. Acute stimulation of EGF-treated cultures with TSH (10 mU/ml) or forskolin (50 microM) caused, as in controls, an increase in apical, but not basal, 125I- efflux within minutes; the peak value of stimulated apical efflux was 10-fold over the prestimulatory level of basoapical FTE 125I- in the same culture. Moreover, the steady state level of basoapical FTE 125I- after the transient efflux peak was higher than that before stimulation and, in fact, approached the corresponding flux in untreated cells. In contrast, in control and TSH-pretreated cultures, the 125I- efflux peak was less pronounced, and the pre- and poststimulatory levels of basoapical FTE 125I- were about equal. FTE 125I- in the apicobasal direction was always low and unresponsive to acute stimulation regardless of pretreatment. In conclusion, EGF-treated porcine thyroid epithelial cells in Transwell culture, despite being released from contact inhibition of growth, are able to concentrate I- and release their I- content in response to acute TSH stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of thyroid-stimulating hormone and phorbol ester on glycosaminoglycan synthesis in porcine thyroid epithelial cells in primary culture

The effects of thyroid-stimulating hormone (TSH) and a tumor promoter: 12-0-tetradecanoyl-phorbol-13-acetate on glycosaminoglycan (GAG) synthesis were studied in porcine thyroid epithelial cells in primary culture. TSH is known to involve cyclic AMP mechanism and phorbol ester to act by protein kinase C pathway. Chronic treatment of cells with TSH increased the synthesis of heparan sulphate associated with the cell layer and hyaluronic acid in the culture medium. Phorbol ester increased the radioactivity of total GAGs in the culture medium but had no effect on GAGs associated with the cell layer. It inhibited the positive effect of TSH on heparan sulphate synthesis. These results suggest that in thyroid epithelial cells the synthesis of the GAGs associated with the cell layer and those secreted into the culture medium are regulated by different intracellular mechanisms.

Inter- and Intracellular luminal formation in porcine thyroid tissues cultured in a collagen substrate.

Porcine thyroid epithelial cells, isolated and cultured on a type I collagen substrate, formed a monolayered sheet. When the apical surface of the monolayer was further covered with the same collagen substrate, the epithelial cells migrated and formed intercellular follicular lumen-like structures. In addition, a true intracellular follicular lumen sometimes appeared, especially in cells of the monolayer region. It was proved by serial sections that the intracellular follicular lumina, 1-5 microns in diameter, lined by rather long microvilli and stained for PAS, had no openings to the extracellular space. The intracellular lumina are presumed to fuse with each other to form an intercellular follicular lumen, or fuse with the performed intercellular follicular lumen. Auto-radiographic studies using 125I revealed that iodination of thyroglobulin takes place in the peripheral region of both intracellular and intercellular follicular lumina.

Synthesis and apical and basolateral secretion of thyroglobulin by thyroid cell monolayers on permeable substrate: modulation by thyrotropin.

In the thyroid glands, thyroglobulin (Tg) is specifically synthesized by follicular cells and then secreted into the apical lumen where it is concentrated and used as a substrate for thyroid hormone synthesis. The presence of Tg in the circulation has been reported in normal and pathological situations. To determine the domains of the plasma membrane, apical and/or basolateral, involved in Tg secretion, porcine thyroid epithelial cells were cultured as a monolayer on the porous bottom of a culture chamber in which both apical and basal media are independently accessible. Control experiments using labeled Tg ascertained the tightness of the monolayer and showed that within 48 h only 0.2-0.5% of the Tg introduced in the apical medium was transferred through the cell layer into the basal compartment. For kinetic studies of Tg synthesis and secretion, monolayers were cultured for up to 72 h in the presence of 35S-methionine and with or without 100 microU/ml thyrotropin (TSH) in the basal medium. Labeled Tg was measured by double immunoprecipitation and by fluorography of polyacrylamide gel electrophoresis. We showed that 80-95% of total secreted Tg was recovered in the apical medium. The remainder was secreted through the basolateral membranes in the basal medium. The amount of tg secreted into the apical compartment was stimulated two- to threefold by TSH whereas no TSH effect was observed on secretion in the basal compartment. Moreover, measuring apical and basal volumes, we observed a net water flow from the apical to the basal side. It was stimulated threefold by TSH, increasing the Tg concentration in the apical compartment of the stimulated cell layer. During the culture time, the amount of Tg synthesized and secreted was increased by TSH, as was the Tg mRNA content, as determined by the dot-blot hybridization method.

Aminopeptidase N is a marker for the apical pole of porcine thyroid epithelial cells in vivo and in culture.

An aminopeptidase N has been detected by immunofluorescence in the apical plasma membrane of porcine thyroid cells, facing the follicular lumen. Freshly isolated cells obtained by tissue trypsinization, lose their polarity and exhibit a homogeneous enzyme distribution over the whole plasma membrane. In thyrotropin-stimulated cultured cells organized into follicles, the enzyme is localized the apical cell pole. In monolayer cells, on the other hand, the enzyme is distributed over the whole surface facing the medium. In both types of cultures fluorescence is also observed in intracytoplasmic organelles. In vivo, aminopeptidase ia a marker of the apical part of the thyroid plasma membrane, but its in vitro localization depends upon cell differentiation related to the culture conditions.